PROJECTSUMMARY Currentcancerchemotherapeuticstypicallycausesignificantnegativesideeffects;?novelchemotherapeutics withoutsuchsideeffectsarethereforehighlysoughtafter.Thebenzylisoquinolinealkaloidnoscapine,which hasalonghistoryofsafeclinicaluseasanantitussive,iscurrentlybeinginvestigatedforanticancerproperties withfarfewersideeffectsthancurrentchemotherapeuticsandhasbeenprescribedoff-labelforthispurpose. Useofnoscapineasacancerchemotherapeuticishinderedbylowpotency,butsyntheticderivativesof noscapine(noscapinoids),especiallyhalogenatedderivatives,havedemonstratedsignificantlyimproved activities.However,onlyalimitedsetofderivativescanbereadilyproducedsynthetically,andfurtherimproved derivativesmaystillexisttobeexplored.Recently,engineeredstrainsofSaccharomycescerevisiaecapableof biosynthesizingnoscapinehavebeendeveloped,andthisbiosyntheticpathwaycouldbeexpandedvia additionalenzymesinordertoprovidereadyaccesstonoscapinoids,boththosecurrentlyunderinvestigation aswellasnovel,potentiallyimprovedderivatives.Whilehalogenatingenzymes(halogenases)existinnature, nosuchhalogenasesareknowntofunctionalizenoscapineorrelatedcompounds.InthisworkIwillutilize proteinengineeringinordertogenerateengineeredhalogenasescapableoffunctionalizingnoscapineto directlyfurnishhalonoscapinoids.Iwillalsoengineerenzymesinthebiosyntheticpathwayofnoscapineto expandtheirsubstratescopestoallowincorporationofderivatizedsubstratestoaffordaccesstoanadditional rangeofnoscapinoidsnotavailableviachemicalorenzymaticsynthesis.Iwillthenperformpathway engineeringtocombinetheseengineeredenzymeswiththeheterologousnoscapinebiosyntheticpathwayin ordertogeneratehighvaluenoscapinoidsforbiologicalcharacterization.Thiswillultimatelyfurnish biosyntheticroutestoarangeofnoscapinoidswithpotentialanticanceractivityandwilldemonstratetheutility ofcombiningproteinengineeringwithsyntheticbiologyfornoveldrugdevelopmentviacombinatorial biosynthesis.Inaddition,thisproposalwillallowmetoapplyandexpandtheskillsIdevelopedinprotein engineeringduringmygraduateworkwhilereceivingtrainingfrommymentorandcoworkersinpathway engineeringandsyntheticbiologyinyeast.